Research Article |
Corresponding author: Nicolas A. Hazzi ( nicolashazzi@gwu.edu ) Academic editor: Miquel A. Arnedo
© 2021 Nicolas A. Hazzi, Gustavo Hormiga.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hazzi NA, Hormiga G (2021) Morphological and molecular evidence support the taxonomic separation of the medically important Neotropical spiders Phoneutria depilata (Strand, 1909) and P. boliviensis (F.O. Pickard-Cambridge, 1897) (Araneae, Ctenidae). ZooKeys 1022: 13-50. https://doi.org/10.3897/zookeys.1022.60571
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The species of the genus Phoneutria (Ctenidae), also called banana spiders, are considered amongst the most venomous spiders in the world. In this study we revalidate P. depilata (Strand, 1909), which had been synonymized with P. boliviensisis (F.O. Pickard-Cambridge, 1897), using morphological and nucleotide sequence data (COI and ITS-2) together with species delimitation methods. We synonymized Ctenus peregrinoides, Strand, 1910 and Phoneutria colombiana Schmidt, 1956 with P. depilata. Furthermore, we designated Ctenus signativenter Strand, 1910 as a nomen dubium because the exact identity of this species cannot be ascertained with immature specimens, but we note that the type locality suggests that the C. signativenter syntypes belong to P. depilata. We also provide species distribution models for both species of Phoneutria and test hypotheses of niche conservatism under an allopatric speciation model. Our phylogenetic analyses support the monophyly of the genus Phoneutria and recover P. boliviensis and P. depilata as sister species, although with low nodal support. In addition, the tree-based species delimitation methods also supported the separate identities of these two species. Phoneutria boliviensis and P. depilata present allopatric distributions separated by the Andean mountain system. Species distribution models indicate lowland tropical rain forest ecosystems as the most suitable habitat for these two Phoneutria species. In addition, we demonstrate the value of citizen science platforms like iNaturalist in improving species distribution knowledge based on occurrence records. Phoneutria depilata and P. boliviensis present niche conservatism following the expected neutral model of allopatric speciation. The compiled occurrence records and distribution maps for these two species, together with the morphological diagnosis of both species, will help to identify risk areas of accidental bites and assist health professionals to determine the identity of the species involved in bites, especially for P. depilata.
Andes, Maxent, niche conservatism, Phylogenetics, species delimitation
The species of the genus Phoneutria are considered aggressive and amongst the most venomous spiders in the world (
Phoneutria boliviensis (Pickard-Cambridge, 1897) is a widespread species distributed from Central America (Costa Rica) to central South America (Bolivia), found across many types of ecosystems and geographical barriers that commonly divide other taxa (e.g. the Andes mountain system that separates many cis and trans Andean lowland lineages) (
At that time, P. boliviensis was known only to occur in the Amazon region, until Valerio (1983) reported this species in Costa Rica. Later,
During field work in Costa Rica, Panama, Colombia, Ecuador and Peru, and careful examination of museum specimens from these countries, we realize that P. boliviensis can be separated into two distinct species. One trans-Andean species, Phoneutria depilata and the true P. boliviensis (cis-Andean) endemic of the Amazon region. Therefore, in this study we revalidated P. depilata that was synonymized with P. boliviensisis by
The material examined and/or collected belongs to the following museums:
MUSM-ENT Museo de Historia Natural, Lima, Peru (D. Silva);
USNM National Museum of Natural History, Smithsonian Institution, Washington DC, USA (H. Wood);
ZMB Museum für Naturkunde der Humboldt Universität, Berlin, Germany (J. Dunlop).
Specimens were preserved in 95% ethanol. Descriptions and terminology follows
Sampling design. Due to the widespread climatic niche of P. depilata, we sequenced seven specimens from Costa Rica, Ecuador and Panama that were collected from mountain to lowland areas, and from dry to rain forests ecosystems (Table
DNA taxon sampling generated in this study. Letters in the haplotype/allele column indicate if individuals have the same sequence in the COI or ITS-2 markers respectively; * indicate that the sequence is unique.
Species | Code | Country | Locality | Latitude / longitude | COI | ITS-2 | Haplotype/allele | Museum code |
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Kiekie curvipes | GH2776 | Costa Rica | Tirimbina Reserve | 10.4164, -84.1199 | MW598451 | MW599260 | */* |
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Ctenus aff. amphora | GH2779 | Brazil | Roraima | 2.7375, -62.075 | MW599262 | -/* |
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Ctenus datus | GH2778 | Panama | Gamboa | 9.1216, -79.7034 | MW598452 | MW599261 | */* |
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Spinoctenus escalerete | GH2777 | Costa Rica | Las Cruces Biological Station | 8.7845, -82.9597 | MW598442 | MW599254 | */* |
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Phoneutria fera | GH2794 | Ecuador | Liana Lodge | -1.056, -77.524 | MW598443 | MW599255 | */A |
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Phoneutria depilata | GH2793 | Ecuador | Caimito, Esmeraldas | 0.7005, -80.0741 | MW598444 | MW599256 | */A |
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Phoneutria depilata | GH2787 | Panama | Gamboa | 9.1216, -79.7034 | MW598448 | MW599256 | */A |
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Phoneutria depilata | GH2792 | Costa Rica | Tirimbina Reserve | 10.4164, -84.1199 | MW598445 | MW599256 | */A |
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Phoneutria depilata | GH2791 | Costa Rica | Cirenas | 9.7199, -85.2119 | MW598446 | */- |
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Phoneutria depilata | GH2790 | Panama | Puerto Amuelles | 8.2841, -82.8691 | MW598447 | C/- |
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Phoneutria depilata | GH2789 | Costa Rica | San Isidro | 10.0182, -84.0551 | MW598447 | MW599256 | C/A |
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Phoneutria depilata | GH2788 | Panama | Puerto Amuelles | 8.2841, -82.8691 | MW598447 | MW599256 | C/A |
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Phoneutria boliviensis | GH2780 | Peru | ACP Panguana | -9.6137, -74.9352 | MW598450 | MW599258 | B/C |
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Phoneutria boliviensis | GH2781 | Peru | Reserva Nacional Allpahuayo Mishana, Biological Station “José Alvarez Alonso” | -3.9663, -73.4368 | MW598450 | MW599259 | B/B |
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Phoneutria boliviensis | GH2782 | Peru | Reserva Nacional Allpahuayo Mishana, Biological Station “José Alvarez Alonso” | -3.9663, -73.4368 | MW598450 | MW599259 | B/B |
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Phoneutria boliviensis | HG2783 | Peru | Madre de Dios, Finca Las Piedras | -12.2259, -69.1142 | MW598450 | MW599258 | B/C | MUSM-ENT 54118 |
Phoneutria boliviensis | HG2784 | Peru | ACP Panguana | -9.6137, -74.9352 | MW598450 | MW599258 | B/C |
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Phoneutria boliviensis | HG2785 | Peru | ACP Panguana | -9.6137, -74.9352 | MW598449 | MW599259 | A/B |
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Phoneutria boliviensis | GH2786 | Peru | Reserva Nacional Allpahuayo Mishana, Biological Station “José Alvarez Alonso” | -3.9663, -73.4368 | MW598449 | MW599257 | A/* |
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Specimens preserved in 95% ethanol were used for DNA extraction using the Qiagen DNEasy kit. Coxae and femora were used for extractions and the rest of the specimen was preserved as a voucher. Two gene fragments frequently used for species recognition and delimitation in spiders (e.g.,
The best partitioning scheme and substitution models were explored using PartitionFinder 2.1.1 using the ‘‘greedy” search strategy and the correction of the Akaike information criterion (AICc). Four partition schemes were used as input data: first, second and third codon position for COI, and ITS-2 as a whole. Phylogenetic analyses were performed using parsimony (MP), maximum likelihood (ML) and Bayesian inferences (BI). The parsimony analyses were carried out in TNT v. 1.5 (
To measure relationships between haplotypes, we constructed haplotype median-joining networks for each marker using PopArt v1.7 (
The three remaining methods used are tree-based. First, we applied the general mixed Yule coalescent model (GMYC,
Finally, we applied the Bayesian Phylogenetics and Phylogeography software (BPP,
We estimated the distribution of P. boliviensis and P. depilata using the Maxent algorithm (
To mitigate the impact of uneven sampling in our occurrence data, we applied a distance correction by taking only one point within a radius of 10 km. We obtained 19 bioclimatic predictor layers summarizing annual trends, seasonality and extremes in precipitation and temperature at a spatial resolution of 30 arc-seconds (i.e. 1 km2) from the WorldClim database (
We ran the models selecting a logistic output and random seed, and the maximum number of background points maintained at 10,000. To assess model performance, we applied k-fold cross validation procedure splitting the occurrences into training and testing records (70% and 30%, respectively), and replicating this process 15 times. Models were evaluated using the Area Under the Curve Metric (AUC) that compares model results with null expectations using a threshold-independent measure. We average the AUC values obtained in the replicates and created confidence intervals values to assess model significance from random model expectations (AUC > 0.5). In order to make the binary distribution maps, habitat suitability values were converted in presence and absence using the 5th percentile as the threshold value (
To test niche conservatism among these two species, we used the niche similarity test (
The tree topologies of the parsimony, Bayesian and maximum likelihood analyses were congruent in recovering with high support metrics the monophyly of the genus Phoneutria and the three morphologically recognized species: Phoneutria depilata, P. fera and P. boliviensis. Therefore, only the likelihood tree is shown (Fig.
Maximum likelihood phylogenetic tree of the concatenated alignment of COI and ITS-2 markers. PB = posterior probabilities (derived from the Bayesian tree), UB = ultrafast bootstrap (derived from the likelihood tree), and JAC = jackknife (derived from the parsimony tree). Support metrics for nodes with low support (UB and PB < 0.95, and Jac < 70) are not shown. Phoneutria images: P. fera (top), P. boliviensis (center), P. depilata (bottom).
mtDNA haplotype networks (Fig.
The ABGD method indicates four species, separating two specimens of P. boliviensis (GH2782 and GH2783) as a separate species. Instead, the mPTP species delimitation analysis indicated with high support (ASV = 0.99) the delimitation of three morphologically recognizable species. In addition, The GMYC analysis produces the same result. The posterior probabilities for the three species in each model tested in BPP were always many times higher than the alternatives scenarios: 0.97 for deep divergence and large population size, 0.33 for deep divergence and small population size (the next most likely scenario was one species with 0.10), 0.71 for shallow divergence and small population size (the next most likely scenario was two species with 0.14) and 1.00 for shallow divergence and large population size. Thus, three parameter combinations suggest the same number of species.
The Fig.
Distribution models of P. boliviensis and P. depilata presented high performance compared to random expectations (AUC = 0.84 ± 0.10 SD for P. boliviensis and AUC = 0.84 ± 0.06 SD for P. depilata). The distribution model of P. depilata highlighted areas with different levels of suitability across Central and South America (Fig.
In the niche comparison analysis, the 1st and 2nd axis of the PCA‐env explained 53.49% and 14.19% of the variance, respectively (Fig.
Phoneutria fera Perty, 1833.
Ctenus boliviensis: F. O. Pickard-Cambridge, 1897: 80, pl. 3, (female holotype from Madre de Dios, Bolivia, fig. 3a-c (male), The Natural History Museum, London not found; see
Ctenus nigriventroides
Strand, 1907: 426 (female holotype from Bolivia, Museum für Natur und Umwelt der Hansestadt, Lübeck presumed lost; see Eickstedt 1979: 111, and
Ctenus valdehirsutulus
Strand, 1910: 318 (syntypes: female from Sara, W. Bolivia, 60 m, 14 March 1907, J. Steinbach leg., in ZMB 30615; female from Sara, Dpto. Sta. Cruz de la Sierra, Bolivia, 500 m, Steinbach, in ZMB 30616, see
Ctenus nigriventoides: Petrunkevitch, 1911: 475 (only citation of
Ctenus chilesicus Strand, 1915: 128 (female holotype from Chile, 1902, O. Hohenemser leg., in SMF-4557).
Phoneutria boliviensis: Schmidt, 1954: 414; 1956: 28; Bücherl 1968: 188; 1969a: 49;
Phoneutria nigriventroides: Bonnet, 1958: 3621 (in part, only material from Bolivia); Eickstedt 1979: 111.
(herein designated; see comments below). Peru: Male from Madre de Dios, Puerto Maldonado, Finca Las Piedras (12.2259°S, 69.1142°W, 260 m), 20.IX.2019, N. Hazzi coll. (MUSM-ENT 54118).
We have designated a neotype for P. boliviensis in accordance with Article 75 of the International Code of Zoological Nomenclature (ICZN 1999). The type material of Ctenus boliviensis was considered lost after examination of the spider material at the Natural History Museum, London (
The syntypes of Ctenus valderhirsutulus were revised by
Colombia: Caqueta: two males, Universidad de la Amazonia (1.4998°N, 75.6632°W, 240 m) Florencia, 30.VII.2019, N. Hazzi, L. Martínez, and E. Across-Valencia (
Males of P. boliviensis resemble those of P. depilata by the truncated apex of the RTA (Fig.
Phoneutria boliviensis (Finca Las Piedras, Madre de Dios, Peru), female genitalia A epigynum, ventral view B vulva, dorsal view. CD = copulatory duct, ELA = epigynal lateral apophysis, ELF = epigynal lateral field, ELG = epigynal lateral guide, EMF = epigynal middle field, FD = fertilization duct, HS = head of spermatheca, PL = posterior lobe, S = spermatheca. Scale bar: 1.00 mm.
Male (from Madre de Dios, Puerto Maldonado, Finca Las Piedras, Peru; MUSM-ENT 54118). Coloration (Figs
Female (from Madre de Dios, Puerto Maldonado, Finca Las Piedras, Peru; MUSM-ENT 054122). Coloration (Figs
Males (n = 6): Total length 9.70–10.60, carapace 4.86–5.90, femur I 5.90–6.72. Females (n = 5): Total length 12.22–15.22, carapace 6.33–6.97, femur I 5.20–5.86.
Lowland tropical rain forests of the Amazon (0–1000 m) in Bolivia, Brazil, Colombia, Ecuador and Peru (Figs
Phoneutria boliviensis is the smallest species of the genus and it inhabits in sympatry with P. fera and P. reidyi.
Ctenus depilatus Strand, 1910. Holotype male from Colombia (ZMB 30615, examined). Valerio 1983: 101, fig. 2 (female).
Ctenus peregrinoides: Strand, 1910: 318 (syntypes: two females from Guatemala, in ZMB 30717, not examined). New synonymy.
Phoneutria depilata: Schmidt, 1954: 417-418.
Phoneutria colombiana Schmidt, 1956: 418; 1956: 28 (female holotype from Colombia, in SMF, not examined). New synonymy.
Phoneutria boliviensis: Simó & Brescovit, 2001: 74 (as senior synonym of P. depilata); Rozwałka, Rutkowski and Bielak-Bielecki 2017: 61, fig. 1b, c (female);
Phoneutria cf. boliviensis: Cathrine & Longhorn, 2017: 13, figs 1–6 (female).
In their revision of Phoneutria
Nicaragua: Región Autónoma de la Costa Caribe Sur: one female, Escondido River (12.1065°N, 84.0256°W, 10 m), 12.VII.1892, C.W. Richmond (USNM). Panama: Panama: one male, Pearls Island, San José (8.270219°N, -79.112038°W, 30 m), 02.IV.1944, J.P. Morrinson (USNM); Bocas del Toro: one female, Changinola, El Silencio (9.3845°N, 82.5356°W, 20 m), E. Marrango (USNM), one male and one female, Gamboa (9.1176°N. 79.6959°W, 50 m), 05.XIII.2018, N. Hazzi and S. Maneses (
Males of P. depilata resemble those of P. boliviensis by the truncated apex of the RTA (Fig.
Phoneutria depilata (from Puerto Amuelles, Chiriquí, Panama), female genitalia A epigynum, ventral view B vulva, dorsal view. CD = copulatory duct, ELA = epigynal lateral apophysis, ELF = epigynal lateral field, ELG = epigynal lateral guide, EMF = epigynal middle field, FD = fertilization duct, HS = head of spermatheca, PL = posterior lobe, S = spermatheca. Scale bars: 1.00 mm.
Male (from Puerto Amuelles, Chiriquí, Panama,
A, B ventral view of the male palp of P. boliviensis (from Pucallpa, Peru) and P. depilata (from Gamboa, Panama), respectively C, D retrolateral tibia apophysis of P. boliviensis and P. depilata, respectively. Scales bars: 0.10 mm (A); 0.20 mm (B); 0.10 mm (C); 0.05 mm (D). C = conductor, E = embolus, IB = internal bulge, LL = locking lobes, MA = median apophysis, ST = subtegulum.
Female (from Puerto Amuelles, Chiriquí, Panama, (
A, B epigynum and vulva (dorsal view) of Phoneutria boliviensis (from Pucallpa, Peru) C, D epigynum and vulva (dorsal view) of Phoneutria depilata (from Caimito, Esmeraldas, Ecuador). ELA = epigynal lateral apophysis, EMF = epigynal middle field, FD = fertilization duct, HS = head of spermatheca, PL = posterior lobe, S = spermatheca. Scales bars: 0.20 mm (A), 0.20 mm (B), 0.30 mm (C), 0.30 mm (D).
Males (n = 5): Total length 21.00–26.37, carapace 11.26–13.75, femur I 13.27–15.84. Females (n = 8): Total length 25.77–34.00, carapace 12.56–15.00, femur I 13.43–14.36.
Trans-Andean region (0–1700 m) in Ecuador, Colombia, Panama, Costa Rica, Nicaragua, Honduras and Guatemala.
This species is found in disturbed habitats associated with both dry and humid tropical forests (0–1700 m), usually on the ground with sparse litter and low vegetation (
Equivalence and similarity tests in environmental space for P. boliviensis and P. depilata A PCA of ecological climatic B the variables contribution to the analyses C graphical results of the equivalency and similarity permutation tests comparing the two species of Phoneutria. Line marks and filled squares are the available environment in each range (M) and occupied space by each species, respectively. Occupied climatic niche by P. boliviensis, P. depilata and niche overlap (D) are in green, red and blue colors, respectively.
Despite the medical importance of Phoneutria, its taxonomy and systematics have been always debated and there is still disagreement about the exact number of species in the genus. For instance, the last two taxonomic revisions of the genus contradict the boundaries of some species.
Previous works of DNA barcoding in Lycosoidea have shown a range of genetic distances among congeneric species of 4–6.9% (
The distance-based method (ABGD) split P. boliviensis into two species, one which was not monophyletic. Several species delimitation studies in spiders have also shown that the ABGD method is sensitive to sampling and tends to over-split species when compared with other methods (
Citizen science platforms have provided unprecedented access to documenting species diversity and distribution across the world (
Phoneutria boliviensis and P. depilata live in lowland areas, and sometimes premontane ecosystems as well (Valerio 1983;
Phylogenetic niche conservatism has been suggested as one of the potential forces in speciation and species richness patterns in the tropics (e.g.,
In conclusion, using morphological and molecular data, together with species delimitation methods our study revalidates Phoneutria depilata as a valid species separate from P. boliviensis. Both species have allopatric distributions separated by the Andean mountains, and species distribution models indicated lowland tropical rain forest ecosystems as the most suitable environments for these species. In addition, this work demonstrated the value of citizen science platforms like iNaturalist for occurrence records and improving species distribution knowledge. Phoneutria depilata and the three Amazonian species presented niche conservatism following the expected neutral model of allopatric speciation. Finally, the morphological diagnosis of these two species and the distribution maps provided in this work will be useful for future studies in venom, epidemiology of bites, and systematics of this venomous groups of spiders.
We thank the Department of Biological Sciences of The George Washington University, the Harlan Fellowship, the Explorers Club Washington DC group and The Early Career Grants of National Geographic for financial support. We also thank the Ernst Mayr Travel Grants (
Concatenated alignment
Data type: DNA alignment
Explanation note: Concatenated DNA Alignment of ITS-2 and COI.
Curated iNaturalist database for Phoneutria boliviensis and P. depilata
Data type: Occurences
Explanation note: Curated iNaturalist database for Phoneutria boliviensis and P. depilata. Accessed in July 2020.